We will extend our membrane and electron-flow studies of mitochondrial P-450's, and study electron flow into the iron-sulfur-siroheme (sulfite and nitrite) reductases. 1. The Cholesterol Side Chain-Cleavage System of Adrenal Mitochondria. This system, consisting of flavoprotein, Fe2-S2 protein and membrane-bound reaction-specific P-450scc, operates via a "shuttle" which delivers electrons to the hydroxylase. We will now examine the final reactions: the addition of the "second electron" to the substrate-oxyferro enzyme and the dissociation of hydroxylated product and ferric P-450. We are able to prepare and examine substrate-stabilized oxyferro complexes. 2. The 11-B Hydroxylation System of Adrenal Mitochondria. This system uses the electron carriers of the side-chain cleavage system, but has its own specific P-450. We wish to study the role of the membrane, the equilibria and kinetics of the P-450's reactions with oxygen and carbon monoxide, and the fate of oxygenated intermediates. Utilizing variants of optical and EPR techniques used in P-450scc, we will try to ascertain the arrangement of the P-450 and its substrates in the membrane. 3. 25-Hydroxy Vitamin D3-1 Hydroxylase of Kidney Mitochondria. This P-450 hydroxylase, the final enzyme in the biosynthesis of the Vitamin D "hormone", has received little mechanistic attention since its original description. We wish to initiate studies on this system, another example of a reaction-specific membrane-bound P-450 supplied by a flavoprotein-iron-sulfur system. We wish to study its arrangement in the membrane, its mechanism of hydroxylation, and possible loci of regulation. 4. Electron Transfer Into Iron-Sulfur-Siroheme Enzymes. Nitrite reductase of spinach appears to be "fed" by ferredoxin; the hemoprotein subunit of the sulfite reductase of entrobacteria receives electrons from a flavoprotein moiety, whose FAD-FMN composition presents the only known analogy to the flavoprotein P-450 reductase of microsomes. We wish to study the details of the complexation between the flavoprotein and hemoprotein, and of electron flow into the siroheme enzyme either from flavins or from ferredoxin.